Methods for making polysaccharide-protein conjugates
AssigneeMerck Sharp & Dohme Corp.
InventorsJitin Bajaj, Michael Albert Winters, Emily Wen, Jian He
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Abstract
The present invention provides methods for making polysaccharide-protein conjugates in which polysaccharides, typically from bacteria, are conjugated to a carrier protein by reductive amination under conditions which improve conjugation reaction consistency, increase consumption of protein during conjugation reaction, generate conjugates of higher molecular weight, and/or reduce the levels of free cyanide in the conjugate reaction product. The polysaccharide-protein conjugates obtained using these methods are useful for inclusion in multivalent vaccines.
Claims34
A method for preparing a polysaccharide protein conjugate by reductive amination, the method comprising:
- a) reacting an activated polysaccharide with a carrier protein in an aqueous buffer in the presence of a cyanoborohydride salt and nickel to form a conjugate reaction mixture;
- b) optionally, adding a strong reducing agent;
- and
- c) purifying the conjugate reaction mixture to remove residual reactant including free cyanide ions.
A method for preparing a Streptococcus pneumoniae serotype 3 polysaccharide protein conjugate by reductive amination, the method comprising:
- a) reacting an activated serotype 3 polysaccharide with a carrier protein in an aqueous buffer in the presence of a cyanoborohydride salt at a temperature in a range from 2 to 25° C. and a pH of 7.0 or less, or a range from 2 to 10° C. and a pH of 7.7 or less to form a conjugate reaction mixture;
- b) optionally, adding a strong reducing agent;
- and
- c) purifying the conjugate reaction mixture to remove residual reactant including free cyanide ions.
A method for activating a Streptococcus pneumoniae serotype 3 polysaccharide by periodate oxidation, the method comprising reacting a serotype 3 polysaccharide with a periodate in an aqueous buffer at a pH in the range from 4.3-6.9 at a temperature in the range from 2-23° C., at a pH in the range from 4.3-7.7 at a temperature in the range from 2-10° C., or at a pH in the range from 4.3-6.2 at a temperature in the range from 2-35° C.
Dependencies are inferred from the claim text.